Human pinch protein homolog

ABSTRACT

The invention provides a human PINCH protein homolog (PINCH-PH) and polynucleotides which identify and encode PINCH-PH. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of PINCH-PH.

This application is a divisional application of U.S. application Ser.No. 09/008,465, filed Jan. 16, 1998, now U.S. Pat. No. 6,008,625.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of ahuman PINCH protein homolog and to the use of these sequences in thediagnosis, treatment, and prevention of cancer and reproductivedisorders.

BACKGROUND OF THE INVENTION

LIM proteins are a family of proteins that share a common structuraldomain. The LIM motif is so named because it was first described inthree proteins from Drosophila melanogaster designated L, I, and M. TheLIM motif is a cysteine-rich region with a characteristic pattern:[C-X-X-C-X_(17±1)-H-X-X-C]-X-X-[C-X-X-C-X_(17±1)-C-X-X-C]. LIM motifsform two loop structures, and coordinate a zinc ion within each loop.

The LIM motif has been identified in a variety of proteins, includingtranscription factors, cytoskeletal proteins, and signaling molecules.LIM proteins are involved in cell fate determination, growth regulation,and oncogenesis. At least twenty-three members of the LIM family havebeen described, from nematodes to humans. Some LIM proteins consist ofone, two, or three repeats of the LIM motif (LIM-only proteins). Otherscontain a LIM motif with a homeodomain (LIM-HD proteins) or a proteinkinase domain (LIM-PK). LIM-PK inhibits the Ras oncogene-mediateddifferentiation of neural PC12 cells. LIM-HD proteins interact with DNAas well as bind to other proteins and are implicated in the control ofdifferentiation of specific cell types. Studies in C. elegansdemonstrated that LIM-HD proteins are involved in control of celldifferentiation. Lin-11, a LIM-HD protein, controls the asymmetric celldivisions during vulval development, while Mec-3 is required for thedifferentiation of mechanosensory neurons. (Way, J. C. and Chalfie, M.(1988) Cell 54:5-16; and Freyd, G. et al (1990) Nature 344:876-879.)

The LIM-only proteins have not been shown to bind DNA, although the LIMstructure is similar to the zinc finger, a well-characterizedDNA-binding domain. LIM-only proteins include the rat cysteine-richintestinal protein (CRIP), the human RBTN1 and RBTN2 proteins, and thechicken zyxin protein. (Higuchi, O. et al (1997) Oncogene 14:1819-1825;Sanchez-Garcia, I. and Rabbitts, T. H. (1994) Trends Genet. 10:315-320;and Dawid, I. B. et al (1995) C.R. Acad. Sci. III 318:295-306.) Thegenes for RBTN1 and RBTN2 are located on chromosome 11. Translocationmutations of chromosome 11 are associated with specific human T-cellacute leukemias. Transgenic expression of RBTN1 or RBTN2 producesleukemia and lymphoma in mice. (McGuire, E. A. et al (1992) Mol. Cell.Biol. 12:4186-4196; Fisch, P. et al (1992) Oncogene 7:2389-2397.)

A LIM-only protein known as PINCH protein (particularly interesting newCys-His protein) was recently cloned from a human fetal liver library.PINCH protein contains five repeats of the LIM motif. Messenger RNA forPINCH protein is widely expressed, particularly in reproductive tissues,heart, and peripheral blood leukocytes. (Rearden, A. (1994) Biochem.Biophys. Res. Commun. 201: 1124-1131).

The discovery of a new human PINCH protein homolog and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, treatment, andprevention of cancer and reproductive disorders.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, human PINCHprotein homolog (PINCH-PH), comprising a sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1.

The invention further provides a substantially purified variant ofPINCH-PH having at least 90% amino acid identity to the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1. The invention also provides anisolated and purified polynucleotide encoding the polypeptide comprisingthe sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The inventionalso includes an isolated and purified polynucleotide variant having atleast 90% polynucleotide identity to the polynucleotide encoding thepolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1.

Additionally, the invention provides a composition comprising apolynucleotide encoding the polypeptide comprising the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1. The invention further provides anisolated and purified polynucleotide which hybridizes under stringentconditions to the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as anisolated and purified polynucleotide which is complementary to thepolynucleotide encoding the polypeptide comprising the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides an isolated and purified polynucleotidecomprising a sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, andan isolated and purified polynucleotide variant having at least 90%polynucleotide identity to the polynucleotide comprising the sequence ofSEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides anisolated and purified polynucleotide which is complementary to thepolynucleotide comprising the sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect,the expression vector is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising a sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, themethod comprising the steps of: (a) culturing the host cell containingan expression vector containing at least a fragment of a polynucleotideencoding PINCH-PH under conditions suitable for the expression of thepolypeptide; and (b) recovering the polypeptide from the host cellculture.

The invention also provides a pharmaceutical composition comprising asubstantially purified PINCH-PH having the sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1 in conjunction with a suitable pharmaceuticalcarrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, as well as a purified agonist and a purified antagonist of thepolypeptide.

The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of PINCH-PH,

The invention also provides a method for treating or preventing areproductive disorder, the method comprising administering to a subjectin need of such treatment an effective amount of an antagonist ofPINCH-PH.

The invention also provides a method for detecting a polynucleotideencoding PINCH-PH in a biological sample containing nucleic acids, themethod comprising the steps of: (a) hybridizing the complement of thepolynucleotide encoding the polypeptide comprising the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleicacids of the biological sample, thereby forming a hybridization complex;and (b) detecting the hybridization complex, wherein the presence of thehybridization complex correlates with the presence of a polynucleotideencoding PINCH-PH in the biological sample. In one aspect, the nucleicacids of the biological sample are amplified by the polymerase chainreaction prior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, and 1E show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of PINCH-PH. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering.,South San Francisco, Calif.).

FIGS. 2A and 2B show the amino acid sequence alignments between PINCH-PH(3540806; SEQ ID NO:1) and PINCH protein (GI 516012; SEQ ID NO:3),produced using the multisequence alignment program of LASERGENE software(DNASTAR Inc, Madison Wis.).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Definitions

“PINCH-PH,” as used herein, refers to the amino acid sequences ofsubstantially purified PINCH-PH obtained from any species, particularlya mammalian species, including bovine, ovine, porcine, murine, equine,and preferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term “agonist,” as used herein, refers to a molecule which, whenbound to PINCH-PH, increases or prolongs the duration of the effect ofPINCH-PH. Agonists may include proteins, nucleic acids, carbohydrates,or any other molecules which bind to and modulate the effect ofPINCH-PH.

An “allele” or an “allelic sequence,” as these terms are used herein, isan alternative form of the gene encoding PINCH-PH. Alleles may resultfrom at least one mutation in the nucleic acid sequence and may resultin altered mRNAs or in polypeptides whose structure or function may ormay not be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toalleles are generally ascribed to natural deletions, additions, orsubstitutions of nucleotides. Each of these types of changes may occuralone, or in combination with the others, one or more times in a givensequence.

“Altered” nucleic acid sequences encoding PINCH-PH, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same PINCH-PHor a polypeptide with at least one functional characteristic ofPINCH-PH. Included within this definition are polymorphisms which may ormay not be readily detectable using a particular oligonucleotide probeof the polynucleotide encoding PINCH-PH, and improper or unexpectedhybridization to alleles, with a locus other than the normal chromosomallocus for the polynucleotide sequence encoding PINCH-PH. The encodedprotein may also be “altered,” and may contain deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent PINCH-PH. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological orimmunological activity of PINCH-PH is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid,positively charged amino acids may include lysine and arginine, andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine; andphenylalanine and tyrosine.

The terms “amino acid” or “amino acid sequence,” as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments”, “immunogenic fragments”, or“antigenic fragments” refer to fragments of PINCH-PH which arepreferably about 5 to about 15 amino acids in length and which retainsome biological activity or immunological activity of PINCH-PH. Where“amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms are not meant to limit the amino acid sequenceto the complete native amino acid sequence associated with the recitedprotein molecule.

“Amplification,” as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,pp. 1-5.)

The term “antagonist,” as it is used herein, refers to a molecule which,when bound to PINCH-PH, decreases the amount or the duration of theeffect of the biological or immunological activity of PINCH-PH.Antagonists may include proteins, nucleic acids, carbohydrates,antibodies, or any other molecules which decrease the effect ofPINCH-PH.

As used herein, the term “antibody” refers to intact molecules as wellas to fragments thereof, such as Fa, F(ab′)₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindPINCH-PH polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to a specificnucleic acid sequence. The term “antisense strand” is used in referenceto a nucleic acid strand that is complementary to the “sense” strand.Antisense molecules may be produced by any method including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes and to block either transcription or translation. Thedesignation “negative” can refer to the antisense strand, and thedesignation “positive” can refer to the sense strand.

As used herein, the term “biologically active,” refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic PINCH-PH, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” such that onlysome of the nucleic acids bind, or it may be “complete,” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding PINCH-PH orfragments of PINCH-PH may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS), and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

The phrase “consensus sequence,” as used herein, refers to a nucleicacid sequence which has been resequenced to resolve uncalled bases,extended using XL-PCR Kit (PE Biosystems, Foster City, Calif.) in the 5′and/or the 3′ direction, and resequenced, or which has been assembledfrom the overlapping sequences of more than one Incyte Clone using acomputer program for fragment assembly, such as the GELVIEW fragmentassembly system (GCG, Madison, Wis.). Some sequences have been bothextended and assembled to produce the consensus sequence.

As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding PINCH-PH,by northern analysis is indicative of the presence of nucleic acidsencoding PINCH-PH in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding PINCH-PH.

A “deletion,” as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term “derivative,” as used herein, refers to the chemicalmodification of PINCH-PH, of a polynucleotide sequence encodingPINCH-PH, or of a polynucleotide sequence complementary to apolynucleotide sequence encoding PINCH-PH. Chemical modifications of apolynucleotide sequence can include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A derivative polynucleotideencodes a polypeptide which retains at least one biological orimmunological function of the natural molecule. A derivative polypeptideis one modified by glycosylation, pegylation, or any similar processthat retains at least one biological or immunological function of thepolypeptide from which it was derived.

The term “homology,” as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology. Theword “identity” may substitute for the word “homology.” A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of reduced stringency. This is not tosay that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% homology oridentity). In the absence of non-specific binding, the substantiallyhomologous sequence or probe will not hybridize to the secondnon-complementary target sequence.

The phrases “percent identity” or “% identity” refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (LASERGENE softwarepackage, DNASTAR). The MEGALIGN program can create alignments betweentwo or more sequences according to different methods, e.g., the ClustalMethod. (Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) TheClustal algorithm groups sequences into clusters by examining thedistances between all pairs. The clusters are aligned pairwise and thenin groups. The percentage similarity between two amino acid sequences,e.g., sequence A and sequence B, is calculated by dividing the length ofsequence A, minus the number of gap residues in sequence A, minus thenumber of gap residues in sequence B, into the sum of the residuematches between sequence A and sequence B, times one hundred. Gaps oflow or of no homology between the two amino acid sequences are notincluded in determining percentage similarity. Percent identity betweennucleic acid sequences can also be calculated by the Clustal Method, orby other methods known in the art, such as the Jotun Hein Method. (See,e.g., Hein, J. (1990) Mehtods Enzymol. 183:626-645.) Identity betweensequences can also be determined by other methods known in the art,e.g., by varying hybridization conditions.

“Human artificial chromosomes” (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat. Genet. 15:345-355.)

The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

“Hybridization,” as the term is used herein, refers to any process bywhich a strand of nucleic acid bonds with a complementary strand throughbase pairing.

The term “hybridization complex” as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution (e.g., C₀t or R₀t analysis) or formed between onenucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid support (e.g., paper, membranes,filters, chips, pins or glass slides, or any other appropriate substrateto which cells or their nucleic acids have been fixed).

The words “insertion” or “addition,” as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term “microarray,” as used herein, refers to an array of distinctpolynucleotides or oligonucleotides arrayed on a substrate, such aspaper, nylon or any other type of membrane, filter, chip, glass slide,or any other suitable solid support.

The term “modulate,” as it appears herein, refers to a change in theactivity of PINCH-PH. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of PINCH-PH.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to an oligonucleotide, nucleotide, polynucleotide, or any fragmentthereof, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In this context, “fragments” refers to those nucleicacid sequences which are greater than about 60 nucleotides in length,and most preferably are at least about 100 nucleotides, at least about1000 nucleotides, or at least about 10,000 nucleotides in length.

The terms “operably associated” or “operably linked,” as used herein,refer to functionally related nucleic acid sequences. A promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the transcription of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in reading frame, certain genetic elements, e.g.,repressor genes, are not contiguously linked to the encoded polypeptidebut still bind to operator sequences that control expression of thepolypeptide.

The term “oligonucleotide,” as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimers,”“primers,” “oligomers,” and “probes,” as these terms are commonlydefined in the art.

“Peptide nucleic acid” (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA and RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

The term “sample,” as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encodingPINCH-PH, or fragments thereof, or PINCH-PH itself may comprise a bodilyfluid; an extract from a cell, chromosome, organelle, or membraneisolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution orbound to a solid support; a tissue; a tissue print; etc.

As used herein, the terms “specific binding” or “specifically binding”refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein recognized by thebinding molecule (i.e., the antigenic determinant or epitope). Forexample, if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term “stringent conditions” refers to conditionswhich permit hybridization between polynucleotide sequences and theclaimed polynucleotide sequences. Suitably stringent conditions can bedefined by, for example, the concentrations of salt or formamide in theprehybridization and hybridization solutions, or by the hybridizationtemperature, and are well known in the art. In particular, stringencycan be increased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS,and 200 μg/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term “substantially purified,” as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A “substitution,” as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, and refers to cells which transiently express the insertedDNA or RNA for limited periods of time.

A “variant” of PINCH-PH, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties (e.g., replacement of leucinewith isoleucine). More rarely, a variant may have “nonconservative”changes (e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE Software (DNASTAR).

The Invention

The invention is based on the discovery of a new human PINCH proteinhomolog (PINCH-PH), the polynucleotides encoding PINCH-PH, and the useof these compositions for the diagnosis, treatment, or prevention ofcancer and reproductive disorders.

Nucleic acids encoding the PINCH-PH of the present invention were firstidentified in Incyte Clone 3540806 from the seminal vesicle cDNA library(SEMVNOT04) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones3540806 (SEMVNOT04), 853536 (NGANNOT01), 2190641 (THYRTUT03), 776025(COLNNOT05), 1703222 (DUODNOT02), 1722945 (BLADNOT060, and 1262471(SYNORAT05).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,1D, and 1E. PINCH-PH is 341 amino acids in length and has two LIM domainsignature sequences, from C₁₅ through F₅₀ and C₇₆ through L₁₀₉. Inaddition, PINCH-PH has a potential amidation site at E₅₉, threepotential glycosylation sites at N₅, N₁₆₅, and N₂₅₉, and a total ofeight potential phosphorylation sites: a cAMP- and cGMP-dependentprotein kinase phosphorylation site at S₃₂₄, three casein kinase IIphosphorylation sites at S₂₃, S₃₁, and S₇₈, three protein kinase Cphosphorylation sites at T₂₉₁, T₃₂₇, and S₃₂₈, and a tyrosine kinasephosphorylation site at Y₅₆. As shown in FIGS. 2A and 2B, PINCH-PH haschemical and structural homology with human PINCH (GI 516012; SEQ IDNO:3). In particular, PINCH-PH and human PINCH share 84% identity. Inaddition, PINCH-PH and human PINCH share the two LIM domain signaturesequences as well as a potential amidation site and two potentialPhosphorylation sites. Northern analysis shows the expression of thissequence in reproductive, gastrointestinal, and nervous systemlibraries, at least 67% of which are immortalized or cancerous and atleast 20% of which involve inflammation and the immune response. Ofparticular note is the expression of PINCH-PH in tumors of the prostate,uterus, bladder, ileum, colon, brain and ganglion.

The invention also encompasses PINCH-PH variants. A preferred PINCH-PHvariant is one which has at least about 80%, more preferably at leastabout 90%, and most preferably at least about 95% amino acid sequenceidentity to the PINCH-PH amino acid sequence, and which contains atleast one functional or structural characteristic of PINCH-PH.

The invention also encompasses polynucleotides which encode PINCH-PH. Ina particular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes aPINCH-PH.

The invention also encompasses a variant of a polynucleotide sequenceencoding PINCH-PH. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding PINCH-PH. A particular aspect ofthe invention encompasses a variant of SEQ ID NO:2 which has at leastabout 80%, more preferably at least about 90%, and most preferably atleast about 95% polynucleotide sequence identity to SEQ ID NO:2. Any oneof the polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of PINCH-PH.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding PINCH-PH, some bearing minimal homology to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring PINCH-PH, and allsuch variations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode PINCH-PH and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring PINCH-PH under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding PINCH-PH or its derivatives possessing a substantiallydifferent codon usage. Codons may be selected to increase the rate atwhich expression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding PINCH-PH and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences which encodePINCH-PH and PINCH-PH derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding PINCH-PH or any fragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, or a fragment of SEQ ID NO:2,under various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; and Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.) Methods for DNA sequencing are well knownand generally available in the art and may be used to practice any ofthe embodiments of the invention. The methods may employ such enzymes asthe Klenow fragment of DNA polymerase I, T7 SEQUENASE DNA ploymerase(Amersham Pharmacia Biotech, Piscataway, N.J.), Taq DNA Polymerase,THERMOSEQUENASE DNA polymerase (Amersham Pharmacia Biotech), orcombinations of polymerases and proofreading exonucleases such as thosefound in the ELONGASE amplification system (Life Technologies,Gaitherburg, Md.). Preferably, the process is automated with machinessuch as the MICRO LAB 2200 System (Hamilton, Reno, Nev.), DNA ENGINEthermal cycler (PTC200; MJ Research, Watertown, Mass.) and the ABICATALYST, ABI PRISM 373 and 377 sequencing systems (PE Biosystems,Foster City, Calif.).

The nucleic acid sequences encoding PINCH-PH may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus. (See, e.g., Sarkar, G. (1993) PCRMethods Applic. 2:318-322.) In particular, genomic DNA is firstamplified in the presence of a primer complementary to a linker sequencewithin the vector and a primer specific to the region predicted toencode the gene. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region. (See, e.g., Triglia, T. etal. (1988) Nucleic Acids Res. 16:8186.) The primers may be designedusing commercially available software such as OLIGO 4.06 Primer analysissoftware (National Biosciences Inc., Plymouth, Minn.) or anotherappropriate program to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to 72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR, which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to place anengineered double-stranded sequence into an unknown fragment of the DNAmolecule before performing PCR. Other methods which may be used toretrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060.) Additionally, one mayuse PCR and Nested Primers to walk genomic DNA. This process avoids theneed to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will include moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR analysis software, PE Biosystems, and the entire process fromloading of samples to computer analysis and electronic data display maybe computer controlled. Capillary electrophoresis is especiallypreferable for the sequencing of small pieces of DNA which might bepresent in limited amounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode PINCH-PH may be used in recombinant DNAmolecules to direct expression of PINCH-PH, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressPINCH-PH.

As will be understood by those of skill in the art, it may beadvantageous to produce PINCH-PH-encoding nucleotide sequencespossessing non-naturally occurring codons. For example, codons preferredby a particular prokaryotic or eukaryotic host can be selected toincrease the rate of protein expression or to produce an RNA transcripthaving desirable properties, such as a half-life which is longer thanthat of a transcript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alterPINCH-PH-encoding sequences for a variety of reasons including, but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding PINCH-PH may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of PINCH-PH activity, it may be usefulto encode a chimeric PINCH-PH protein that can be recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between the PINCH-PH encodingsequence and the heterologous protein sequence, so that PINCH-PH may becleaved and purified away from the heterologous moiety.

In another embodiment, sequences encoding PINCH-PH may be synthesized,in whole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Symp. Ser. (7)215-223,and Horn, T. et al. (1980) Nucl. Acids Symp. Ser. (7)225-232.)Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of PINCH-PH, or a fragmentthereof. For example, peptide synthesis can be performed using varioussolid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science269:202-204.) Automated synthesis may be achieved using the ABI 431APeptide Synthesizer (PE Biosystems).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or by sequencing. (See, e.g., Creighton, T. (1983) Proteins,Structures and Molecular Properties, WH Freeman and Co., New York, N.Y.)Additionally, the amino acid sequence of PINCH-PH, or any part thereof,may be altered during direct synthesis and/or combined with sequencesfrom other proteins, or any part thereof, to produce a variantpolypeptide.

In order to express a biologically active PINCH-PH, the nucleotidesequences encoding PINCH-PH or derivatives thereof may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding PINCH-PH andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding PINCH-PH. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The “control elements” or “regulatory sequences” are thosenon-translated regions, e.g., enhancers, promoters, and 5′ and 3′untranslated regions, of the vector and polynucleotide sequencesencoding PINCH-PH which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters, e.g.,hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla,Calif.) or PSPORT1 Plasmid, (Life Technologies) may be used. Thebaculovirus polyhedrin promoter may be used in insect cells. Promotersor enhancers derived from the genomes of plant cells (e.g., heat shock,RUBISCO, and storage protein genes) or from plant viruses (e.g., viralpromoters or leader sequences) may be cloned into the vector. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses are preferable. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding PINCH-PH, vectorsbased on SV40 or EBV may be used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for PINCH-PH. For example, when largequantities of PINCH-PH are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding PINCH-PH may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced, and pIN vectors. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) PGEX Vectors (AmershamPharmacia Biotech) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters, such as alpha factor, alcoholoxidase, and PGH, may be used. (See, e.g., Ausubel, supra; and Grant etal. (1987) Methods Enzymol. 153:516-544.)

In cases where plant expression vectors are used, the expression ofsequences encoding PINCH-PH may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.)Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews. (See, e.g., Hobbs,S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology(1992) McGraw Hill, New York, N.Y.; pp. 191-196.)

An insect system may also be used to express PINCH-PH. For example, inone such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encodingPINCH-PH may be cloned into a non-essential region of the virus, such asthe polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of sequences encoding PINCH-PH willrender the polyhedrin gene inactive and produce recombinant viruslacking coat protein. The recombinant viruses may then be used toinfect, for example, S. frugiperda cells or Trichoplusia larvae in whichPINCH-PH may be expressed. (See, e.g., Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. 91:3224-3227.)

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding PINCH-PH may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing PINCH-PH in infected host cells. (See, e.g.,Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) Inaddition, transcription enhancers, such as the Rous sarcoma virus (RSV)enhancer, may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding PINCH-PH. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding PINCH-PH and its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularcell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl.Cell Differ. 20:125-162.)

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding,and/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available fromthe American Type Culture Collection (ATCC, Bethesda, Md.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

For long term, high yield production of recombinant proteins, stableexpression is preferred. For example, cell lines capable of stablyexpressing PINCH-PH can be transformed using expression vectors whichmay contain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for about 1 to 2 days in enriched media before being switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase genes and adenine phosphoribosyltransferase genes,which can be employed in tk⁻ or apr⁻ cells, respectively. (See, e.g.,Wigler, M. et al. (1977) Cell 11:223-232; and Lowy, I. et al. (1980)Cell 22:817-823) Also, antimetabolite, antibiotic, or herbicideresistance can be used as the basis for selection. For example, dhfrconfers resistance to methotrexate; npt confers resistance to theaminoglycosides neomycin and G-418; and als or pat confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively. (See,e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570;Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1″14; and Murry,supra.) Additional selectable genes have been described, e.g., trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine. (See,e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.85:8047-8051.) Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, β glucuronidase and itssubstrate GUS, luciferase and its substrate luciferin. Green fluorescentproteins (GFP) (Clontech, Palo Alto, Calif.) are also used (See, e.g.,Chalfie, M. et al. (1994) Science 263:802-805.) These markers can beused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system. (See, e.g., Rhodes, C. A. et al. (1995) Methods Mol.Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingPINCH-PH is inserted within a marker gene sequence, transformed cellscontaining sequences encoding PINCH-PH can be identified by the absenceof marker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding PINCH-PH under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding PINCH-PH and express PINCH-PH may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

The presence of polynucleotide sequences encoding PINCH-PH can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding PINCH-PH.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding PINCH-PHto detect transformants containing DNA or RNA encoding PINCH-PH.

A variety of protocols for detecting and measuring the expression ofPINCH-PH, using either polyclonal or monoclonal antibodies specific forthe protein, are known in the art. Examples of such techniques includeenzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on PINCH-PH is preferred, but a competitivebinding assay may be employed. These and other assays are well describedin the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; and Maddox, D.E. et al. (1983) J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding PINCH-PH includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encodingPINCH-PH, or any fragments thereof, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byAmersham Pharmacia Biotech and Promega (Madison, Wis.). Suitablereporter molecules or labels which may be used for ease of detectioninclude radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding PINCH-PH maybe cultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodePINCH-PH may be designed to contain signal sequences which directsecretion of PINCH-PH through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding PINCH-PH tonucleotide sequences encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences, such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.), between the purificationdomain and the PINCH-PH encoding sequence may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing PINCH-PH and a nucleic acid encoding 6histidine residues preceding a thioredoxin or an enterokinase cleavagesite. The histidine residues facilitate purification on immobilizedmetal ion affinity chromatography. (IMAC) (See, e.g., Porath, J. et al.(1992) Prot. Exp. Purif. 3: 263-281.) The enterokinase cleavage siteprovides a means for purifying PINCH-PH from the fusion protein. (See,e.g., Kroll, D. J. et al. (1993) DNA Cell Biol. 12:441-453.)

Fragments of PINCH-PH may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques. (See, e.g., Creighton, T. E. (1984) Protein: Structures andMolecular Properties, pp. 55-60, W.H. Freeman and Co., New York, N.Y.)Protein synthesis may be performed by manual techniques or byautomation. Automated synthesis may be achieved, for example, using theABI 431A Peptide Synthesizer (PE Biosystems). Various fragments ofPINCH-PH may be synthesized separately and then combined to produce thefull length molecule.

Therapeutics

Chemical and structural homology exists between PINCH-PH and PINCH fromhuman (GI 516012). In addition, PINCH-PH is expressed in reproductive,gastrointestinal, and neural Jan. 8, 1998 tissues. Therefore, PINCH-PHappears to play a role in cancer and reproductive disorders.

Therefore, in one embodiment, an antagonist of PINCH-PH may beadministered to a subject to treat or prevent a cancer. Such a cancermay include, but is not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus. In oneaspect, an antibody which specifically binds PINCH-PH may be useddirectly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress PINCH-PH.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding PINCH-PH may be administered to a subject totreat or prevent a cancer including, but not limited to, those describedabove.

In a further embodiment, an antagonist of PINCH-PH may be administeredto a subject to treat or prevent a reproductive disorder. Such adisorder may include, but is not limited to disorders of prolactinproduction; infertility, including tubal disease, ovulatory defects, andendometriosis; disruptions of the estrous cycle, disruptions of themenstrual cycle, polycystic ovary syndrome, ovarian hyperstimulationsyndrome, endometrial and ovarian tumors, autoimmune disorders, ectopicpregnancy, and teratogenesis; cancer of the breast, fibrocystic breastdisease, and galactorrhea; disruptions of spermatogenesis, abnormalsperm physiology, cancer of the testis, cancer of the prostate, benignprostatic hyperplasia, and prostatitis, carcinoma of the male breast andgynecomastia. In one aspect, an antibody which specifically bindsPINCH-PH may be used directly as an antagonist or indirectly as atargeting or delivery mechanism for bringing a pharmaceutical agent tocells or tissue which express PINCH-PH.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding PINCH-PH may be administered to a subject totreat or prevent a reproductive disorder including, but not limited to,those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of PINCH-PH may be produced using methods which aregenerally known in the art. In particular, purified PINCH-PH may be usedto produce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind PINCH-PH. Antibodies to PINCH-PHmay also be generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith PINCH-PH or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to PINCH-PH have an amino acid sequence consisting ofat least about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of PINCH-PH aminoacids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to PINCH-PH may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce PINCH-PH-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.).

Antibody fragments which contain specific binding sites for PINCH-PH mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between PINCH-PH and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering PINCH-PH epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

In another embodiment of the invention, the polynucleotides encodingPINCH-PH, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding PINCH-PH may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding PINCH-PH. Thus, complementary molecules orfragments may be used to modulate PINCH-PH activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding PINCH-PH.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencescomplementary to the polynucleotides of the gene encoding PINCH-PH.(See, e.g., Sambrook, supra; and Ausubel, supra.)

Genes encoding PINCH-PH can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding PINCH-PH. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector, and may last even longer ifappropriate replication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′, or regulatory regions of the gene encodingPINCH-PH. Oligonucleotides derived from the transcription initiationsite, e.g., between about positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingPINCH-PH.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding PINCH-PH. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist ofPINCH-PH, antibodies to PINCH-PH, and mimetics, agonists, antagonists,or inhibitors of PINCH-PH. The compositions may be administered alone orin combination with at least one other agent, such as a stabilizingcompound, which may be administered in any sterile, biocompatiblepharmaceutical carrier including, but not limited to, saline, bufferedsaline, dextrose, and water. The compositions may be administered to apatient alone, or in combination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of PINCH-PH, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example PINCH-PH or fragments thereof, antibodies ofPINCH-PH, and agonists, antagonists or inhibitors of PINCH-PH, whichameliorates the symptoms or condition. Therapeutic efficacy and toxicitymay be determined by standard pharmaceutical procedures in cell culturesor with experimental animals, such as by calculating the ED50 (the dosetherapeutically effective in 50% of the population) or LD50 (the doselethal to 50% of the population) statistics. The dose ratio oftherapeutic to toxic effects is the therapeutic index, and it can beexpressed as the LD50/ED50 ratio. Pharmaceutical compositions whichexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies are used to formulate a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that includesthe ED50 with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, the sensitivity of the patient,and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind PINCH-PH maybe used for the diagnosis of disorders characterized by expression ofPINCH-PH, or in assays to monitor patients being treated with PINCH-PHor agonists, antagonists, or inhibitors of PINCH-PH. Antibodies usefulfor diagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for PINCH-PH include methodswhich utilize the antibody and a label to detect PINCH-PH in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

A variety of protocols for measuring PINCH-PH, including ELISAs, RIAs,and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of PINCH-PH expression. Normal or standardvalues for PINCH-PH expression are established by combining body fluidsor cell extracts taken from normal mammalian subjects, preferably human,with antibody to PINCH-PH under conditions suitable for complexformation The amount of standard complex formation may be quantitated byvarious methods, preferably by photometric means. Quantities of PINCH-PHexpressed in subject, control, and disease samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingPINCH-PH may be used for diagnostic purposes. The polynucleotides whichmay be used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofPINCH-PH may be correlated with disease. The diagnostic assay may beused to determine absence, presence, and excess expression of PINCH-PH,and to monitor regulation of PINCH-PH levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding PINCH-PH or closely related molecules may be used to identifynucleic acid sequences which encode PINCH-PH. The specificity of theprobe, whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding PINCH-PH,alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe PINCH-PH encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the PINCH-PH gene.

Means for producing specific hybridization probes for DNAs encodingPINCH-PH include the cloning of polynucleotide sequences encodingPINCH-PH or PINCH-PH derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, are commercially available,and may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, by radionuclides such as ³²P or ³⁵S, or byenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

Polynucleotide sequences encoding PINCH-PH may be used for the diagnosisof a disorder associated with expression of PINCH-PH. Examples of such adisorder include, but are not limited to, cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus, and reproductive disorders,such as disorders of prolactin production; infertility, including tubaldisease, ovulatory defects, and endometriosis; disruptions of theestrous cycle, disruptions of the menstrual cycle, polycystic ovarysyndrome, ovarian hyperstimulation syndrome, endometrial and ovariantumors, autoimmune disorders, ectopic pregnancy, and teratogenesis;cancer of the breast, fibrocystic breast disease, and galactorrhea;disruptions of spermatogenesis, abnormal sperm physiology, cancer of thetestis, cancer of the prostate, benign prostatic hyperplasia, andprostatitis, carcinoma of the male breast and gynecomastia. Thepolynucleotide sequences encoding PINCH-PH may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and ELISA-like assays; and inmicroarrays utilizing fluids or tissues from patients to detect alteredPINCH-PH expression. Such qualitative or quantitative methods are wellknown in the art.

In a particular aspect, the nucleotide sequences encoding PINCH-PH maybe useful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingPINCH-PH may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding PINCH-PH inthe sample indicates the presence of the associated disorder. Suchassays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies, in clinical trials, orto monitor the treatment of an individual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of PINCH-PH, a normal or standard profile for expressionis established. This may be accomplished by combining body fluids orcell extracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding PINCH-PH, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding PINCH-PH may involve the use of PCR. These oligomersmay be chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding PINCH-PH, or a fragment of a polynucleotide complementary tothe polynucleotide encoding PINCH-PH, and will be employed underoptimized conditions for identification of a specific gene or condition.Oligomers may also be employed under less stringent conditions fordetection or quantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of PINCH-PHinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;and Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA-like format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

In one embodiment, the microarray is prepared and used according tomethods known in the art. (See, e.g., Chee et al. (1995) PCT applicationWO95/11995; Lockhart, D. J. et al. (1996) Nat. Biotech. 14:1675-1680;and Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619.)

The microarray is preferably composed of a large number of uniquesingle-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs. The oligonucleotidesare preferably about 6 to 60 nucleotides in length, more preferablyabout 15 to 30 nucleotides in length, and most preferably about 20 to 25nucleotides in length. It may be preferable to use oligonucleotideswhich are about 7 to 10 nucleotides in length. The microarray maycontain oligonucleotides which cover the known 5′ or 3′ sequence,sequential oligonucleotides which cover the full length sequence, orunique oligonucleotides selected from particular areas along the lengthof the sequence. Polynucleotides used in the microarray may beoligonucleotides specific to a gene or genes of interest.Oligonucleotides can also be specific to one or more unidentified cDNAsassociated with a particular cell type or tissue type. It may beappropriate to use pairs of oligonucleotides on a microarray. The firstoligonucleotide in each pair differs from the second oligonucleotide byone nucleotide. This nucleotide is preferably located in the center ofthe sequence. The second oligonucleotide serves as a control. The numberof oligonucleotide pairs may range from about 2 to 1,000,000.

In order to produce oligonucleotides for use on a microarray, the geneof interest is examined using a computer algorithm which starts at the5′ end, or, more preferably, at the 3′ end of the nucleotide sequence.The algorithm identifies oligomers of defined length that are unique tothe gene, have a GC content within a range suitable for hybridization,and lack secondary structure that may interfere with hybridization. Inone aspect, the oligomers may be synthesized on a substrate using alight-directed chemical process. (See, e.g., Chee et al., supra. Thesubstrate may be any suitable solid support, e.g., paper, nylon, anyother type of membrane, or a filter, chip, or glass slide.

In another aspect, the oligonucleotides may be synthesized on thesurface of the substrate using a chemical coupling procedure and an inkjet application apparatus. (See, e.g., Baldeschweiler et al. (1995) PCTapplication WO95/251116.) An array analogous to a dot or slot blot(HYBRIDOT Apparatus Life Technologies) may be used to arrange and linkcDNA fragments or oligonucleotides to the surface of a substrate using avacuum system or thermal, UV, mechanical, or chemical bondingprocedures. An array may also be produced by hand or by using availabledevices, materials, and machines, e.g. multichannel pipettors or roboticinstruments. The array may contain from 2 to 1,000,000 or any otherfeasible number of oligonucleotides.

In order to conduct sample analysis using the microarrays,polynucleotides are extracted from a sample. The sample may be obtainedfrom any bodily fluid, e.g., blood, urine, saliva, phlegm, gastricjuices, cultured cells, biopsies, or other tissue preparations. Toproduce probes, the polynucleotides extracted from the sample are usedto produce nucleic acid sequences complementary to the nucleic acids onthe microarray. If the microarray contains cDNAs, antisense RNAs (aRNAs)are appropriate probes. Therefore, in one aspect, mRNA isreverse-transcribed to cDNA. The cDNA, in the presence of fluorescentlabel, is used to produce fragment or oligonucleotide aRNA probes. Thefluorescently labeled probes are incubated with the microarray so thatthe probes hybridize to the microarray oligonucleotides. Nucleic acidsequences used as probes can include polynucleotides, fragments, andcomplementary or antisense sequences produced using restriction enzymes,PCR, or other methods known in the art.

Hybridization conditions can be adjusted so that hybridization occurswith varying degrees of complementarity. A scanner can be used todetermine the levels and patterns of fluorescence after removal of anynonhybridized probes. The degree of complementarity and the relativeabundance of each oligonucleotide sequence on the microarray can beassessed through analysis of the scanned images. A detection system maybe used to measure the absence, presence, or level of hybridization forany of the sequences. (See, e.g., Heller, R. A. et al. (1997) Proc.Natl. Acad. Sci. 94:2150-2155.)

In another embodiment of the invention, nucleic acid sequences encodingPINCH-PH may be used to generate hybridization probes useful in mappingthe naturally occurring genomic sequence. The sequences may be mapped toa particular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial PI constructions, or single chromosomecDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding PINCH-PH on a physical chromosomal map anda specific disorder, or a predisposition to a specific disorder, mayhelp define the region of DNA associated with that disorder. Thenucleotide sequences of the invention may be used to detect differencesin gene sequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., AT to 11q22-23, any sequences mapping to that area may representassociated or regulatory genes for further investigation. (See, e.g.,Gatti, R. A. et al. (1988) Nature 336:577-580.) The nucleotide sequenceof the subject invention may also be used to detect differences in thechromosomal location due to translocation, inversion, etc., amongnormal, carrier, or affected individuals.

In another embodiment of the invention, PINCH-PH, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenPINCH-PH and the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with PINCH-PH, orfragments thereof, and washed. Bound PINCH-PH is then detected bymethods well known in the art. Purified PINCH-PH can also be coateddirectly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding PINCH-PH specificallycompete with a test compound for binding PINCH-PH. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with PINCH-PH.

In additional embodiments, the nucleotide sequences which encodePINCH-PH may be used in any molecular biology techniques that have yetto be developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I. SEMVNOT04 cDNA Library Construction

The seminal vesicle cDNA library was constructed using tissue isolatedfrom a 61-year-old Caucasian male during a radical prostatectomy.Pathology indicated the seminal vesicles were negative for tumor.Pathology for the associated tumor tissue indicated adenocarcinoma,Gleason grade 3+3, forming a predominant mass involving the right sidecentrally and peripherally. The tumor invaded the right mid-posteriorcapsule but did not extend beyond it. The patient presented withinduration, hyperplasia of the prostate, and elevated prostate specificantigen. Patient history included renal failure, osteoarthritis, leftrenal artery stenosis, benign hypertension, thrombocytopenia, andhyperlipidemia. The frozen tissue was homogenized and lysed in TRIZOLreagent (1 g tissue/10 ml TRIZOL; Life Technologies) using a POLYTRONhomogenizer (PT-3000; (Brinkmann Instruments, Westbury, N.Y.). After abrief incubation on ice, chloroform was added (1:5 v/v) and the lysatewas centrifuged. The upper chloroform layer was removed to a fresh tubeand the RNA extracted with isopropanol, resuspended in DEPC-treatedwater, and treated with DNase for 25 min at 37° C. The RNA wasre-extracted twice with acid phenol-chloroform pH 4.7 and precipitatedusing 0.3M sodium acetate and 2.5 volumes ethanol. The mRNA was thenisolated using the OLIGOTEX Kit (Qiagen Valencia, Calif.), and cDNAsynthesis was initiated using a NotI-oligo(dT) primer. cDNA was blunted,ligated to EcoRI adaptors, digested with NotI, and ligated into thepINCY vector (Incyte Pharmaceuticals, Palo Alto, Calif.).

II. Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL PREP96 plasmid Kit (QIAGEN). The recommended protocol was employed exceptfor the following changes: 1) the bacteria were cultured in 1 ml ofsterile Terrific Broth (Life Technologies) with carbenicillin at 25 mg/land glycerol at 0.4%; 2) after inoculation, the cultures were incubatedfor 19 hours and at the end of incubation, the cells were lysed with 0.3ml of lysis buffer; and 3) following isopropanol precipitation, theplasmid DNA pellet was resuspended in 0.1 ml of distilled water. Afterthe last step in the protocol, samples were transferred to a 96-wellblock for storage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94: 441f), using MICROLAB 2200 system (Hamilton) in combinationwith DNA ENGINE thermal cyclers (MJ Research) and sequenced using ABIPRISM 377 sequencing systems (PE Biosystems).

III. Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al.(1990) J. Mol. Biol. 215:403-410.)

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms couldhave been used when dealing with primary sequence patterns and secondarystructure gap penalties. (See, e.g., Smith, T. et al. (1992) ProteinEngineering 5:35-51.) The sequences disclosed in this application havelengths of at least 49 nucleotides and have no more than 12% uncalledbases (where N is recorded rather than A, C, G, or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻⁸ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam),and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for homology.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; andAusubel, F. M. et al. supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ database (Incyte Pharmaceuticals). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or homologous.

The basis of the search is the product score, which is defined as:

sequence identity×% maximum BLAST score/100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact.Homologous molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding PINCH-PH occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of PINCH-PH Encoding Polynucleotides

The nucleic acid sequence of Incyte Clone 3540806 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension of anantisense polynucleotide, and the other was synthesized to initiateextension of a sense polynucleotide. Primers were used to facilitate theextension of the known sequence “outward” generating ampliconscontaining new unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06 Primeranalysis software (National Biosciences), or another appropriateprogram, to be about 22 to 30 nucleotides in length, to have a GCcontent of about 50% or more, and to anneal to the target sequence attemperatures of about 68° C. to about 72° C. Any stretch of nucleotideswhich would result in hairpin structures and primer-primer dimerizationswas avoided.

Selected human cDNA libraries (Life Technologies) were used to extendthe sequence. If more than one extension is necessary or desired,additional sets of primers are designed to further extend the knownregion.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the DNA ENGINE thermal cycler (MJResearch), beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, with the followingparameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat steps 4 through 6 for anadditional 15 cycles Step 8 94° C. for 15 sec Step 9 65° C. for 1 minStep 10 68° C. for 7:15 min Step 11 Repeat steps 8 through 10 for anadditional 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (andholding)

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using the QIAQUICK Kit Qiagen, and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (L B) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing 2× Carb. Thefollowing day, several colonies were randomly picked from each plate andcultured in 150 μl of liquid LB/2× Carb medium placed in an individualwell of an appropriate commercially-available sterile 96-well microtiterplate. The following day, 5 μl of each overnight culture was transferredinto a non-sterile 96-well plate and, after dilution 1:10 with water, 5μl from each sample was transferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ D NO:2 is used to obtain5′ regulatory sequences using the procedure above, oligonucleotidesdesigned for 5′ extension, and an appropriate genomic library.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 Primer analysis software (National Biosciences) andlabeled by combining 50 pmol of each oligomer, 250 μCi of [γ-³²P]adenosine triphosphate (Amersham Pharmacia Biotech), and T4polynucleotide kinase (NEN Life Science Products, Boston, Mass.). Thelabeled oligonucleotides are substantially purified using a SEPHADEXG-25 superfine resin column (Amersham Pharmacia Biotech). An aliquotcontaining 10⁷ counts per minute of the labeled probe is used in atypical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bgl II, Eco RIPst I, Xba 1, or Pvu II (NEN Life Science Products).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Eastman Kodak Rochester, N.Y.) is exposed to the blots for severalhours, hybridization patterns are compared visually.

VII. Microarrays

To produce oligonucleotides for a microarray, one of the nucleotidesequences of the present invention is examined using a computeralgorithm which starts at the 3′ end of the nucleotide sequence. Foreach, the algorithm identifies oligomers of defined length that areunique to the nucleic acid sequence, have a GC content within a rangesuitable for hybridization, and lack secondary structure that wouldinterfere with hybridization. The algorithm identifies approximately 20oligonucleotides corresponding to each nucleic acid sequence. For eachsequence-specific oligonucleotide, a pair of oligonucleotides issynthesized in which the first oligonucleotides differs from the secondoligonucleotide by one nucleotide in the center of the sequence. Theoligonucleotide pairs can be arranged on a substrate, e.g. a siliconchip, using a light-directed chemical process. (See, e.g., Chee, supra.)

In the alternative, a chemical coupling procedure and an ink jet devicecan be used to synthesize oligomers on the surface of a substrate. (See,e.g., Baldeschweiler, supra.) An array analogous to a dot or slot blotmay also be used to arrange and link fragments or oligonucleotides tothe surface of a substrate using or thermal, UV, mechanical, or chemicalbonding procedures, or a vacuum system. A typical array may be producedby hand or using available methods and machines and contain anyappropriate number of elements. After hybridization, nonhybridizedprobes are removed and a scanner used to determine the levels andpatterns of fluorescence. The degree of complementarity and the relativeabundance of each oligonucleotide sequence on the microarray may beassessed through analysis of the scanned images.

VIII. Complementary Polynucleotides

Sequences complementary to the PINCH-PH-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring PINCH-PH. Although use of oligonucleotidescomprising from about 15 to 30 base pairs is described, essentially thesame procedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 primeranalysis software (National Bioscience) and the coding sequence ofPINCH-PH. To inhibit transcription, a complementary oligonucleotide isdesigned from the most unique 5′ sequence and used to prevent promoterbinding to the coding sequence. To inhibit translation, a complementaryoligonucleotide is designed to prevent ribosomal binding to thePINCH-PH-encoding transcript.

IX. Expression of PINCH-PH

Expression of PINCH-PH is accomplished by subcloning the cDNA into anappropriate vector and transforming the vector into host cells. Thisvector contains an appropriate promoter, e.g., B-galactosidase upstreamof the cloning site, operably associated with the cDNA of interest.(See, e.g., Sambrook, supra, pp. 404-433; and Rosenberg, M. et al.(1983) Methods Enzymol. 101:123-138.)

Induction of an isolated, transformed bacterial strain with isopropylbeta-D-thiogalactopyranoside (IPTG) using standard methods produces afusion protein which consists of the first 8 residues ofβ-galactosidase, about 5 to 15 residues of linker, and the full lengthprotein. The signal residues direct the secretion of PINCH-PH intobacterial growth media which can be used directly in the following assayfor activity.

X. Demonstration of PINCH-PH Activity

The activity of PINCH-PH is determined by its ability to promotedifferentiation of permeabilized C2 muscle cells. The basis of thisassay lies in the ability of LIM-only proteins to substitute for muscleLIM protein (MLP) in promoting the differentiation of mouse C2 myogeniccells. Shifting C2 cells from high serum medium to low-serum mediuminduces differentiation of these cells, wherein they change from roundcells to spindle-shaped cells. In addition, the cells express myotubulesand other cytoskeletal components characteristic of a mature musclecell. C2 cells which have been stably transfected with a vectorexpressing antisense to the MLP message (C2-AS cells) do not undergodifferentiation following a shift to low-serum media. However, thesecells can be induced to undergo differentiation under these conditionsprovided they are permeabilized and exposed to purified MLP ortransiently transfected with a vector expressing MLP. In addition, otherLIM-only proteins including Drosophila homolog of MLP (DMLP) andcysteine-rich intestinal protein (CRIP), are able to substitute for MLPin promoting differentiation of C2-AS cells. Thus, the activity of asample containing PINCH-PH is assayed by determining it's ability topromote differentiation in C2-AS cells. Following permeabilization andtreatment with PINCH-PH-containing samples, the degree ofdifferentiation of C2-AS cells is measured by visual examination, e.g.,scoring the cells for the change in morphology characteristic ofdifferentiated C2-AS cells. (Arber, S. et al (1994) Cell 79:221-231).

XI. Production of PINCH-PH Specific Antibodies

PINCH-PH substantially purified using PAGE electrophoresis (see, e.g.,Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or otherpurification techniques, is used to immunize rabbits and to produceantibodies using standard protocols. The PINCH-PH amino acid sequence isanalyzed using LASERGENE software (DNASTAR Inc) to determine regions ofhigh immunogenicity, and a corresponding oligopeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Methods for selection of appropriate epitopes, such as those near theC-terminus or in hydrophilic regions are well described in the art.(See, e.g., Ausubel et al. supra, ch. 11.)

Typically, the oligopeptides are 15 residues in length, and aresynthesized using an ABI 431A Peptide Synthesizer (PE Biosystems) usingFmoc chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel et al. supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide activity, for example, bybinding the peptide to plastic, blocking with 1% BSA, reacting withrabbit antisera, washing, and reacting with radio-iodinated goatanti-rabbit IgG.

XII. Purification of Naturally Occurring PINCH-PH Using SpecificAntibodies

Naturally occurring or recombinant PINCH-PH is substantially purified byimmunoaffinity chromatography using antibodies specific for PINCH-PH. Animmunoaffinity column is constructed by covalently couplinganti-PINCH-PH antibody to an activated chromatographic resin, such asCNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After thecoupling, the resin is blocked and washed according to themanufacturer's instructions.

Media containing PINCH-PH are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of PINCH-PH (e.g., high ionic strength buffers in thepresence of detergent). The column is eluted under conditions thatdisrupt antibody/PINCH-PH binding (e.g., a buffer of pH 2 to pH 3, or ahigh concentration of a chaotrope, such as urea or thiocyanate ion), andPINCH-PH is collected.

XIII. Identification of Molecules Which Interact with PINCH-PH

PINCH-PH, or biologically active fragments thereof, are labeled with¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529 (hyphen)-539.) Candidate molecules previously arrayed in thewells of a multi-well plate are incubated with the labeled PINCH-PH,washed, and any wells with labeled PINCH-PH complex are assayed. Dataobtained using different concentrations of PINCH-PH are used tocalculate values for the number, affinity, and association of PINCH-PHwith the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

What is claimed is:
 1. A substantially purified human ParticularlyInteresting New Cys-His protein homolog (PINCH-PH) comprising a sequenceof SEQ ID NO:1.
 2. A substantially purified variant of PINCH-PH havingat least 90% amino acid identity to the sequence of claim 1 and whichpromotes differentiation of permeabilized cultured cells.
 3. Acomposition comprising the PINCH-PH of claim 1 in conjunction with acarrier.
 4. A biologically active fragment of the protein of claim 1comprising amino acids C15-F50 or C76-109 of the amino acid sequence ofSEQ ID NO:1 wherein the biological activity promotes differentiation ofpermeabilized cultured cells.
 5. An antigenic fragment of SEQ ID NO:1comprising C15-F50 or C76-L109.
 6. A composition comprising the fragmentof claim
 4. 7. A fusion protein containing the fragment of claim
 4. 8. Amethod for using a protein to screen a library of molecules or compoundsto identify at least one molecule or compound which specifically bindsthe protein, the method comprising: (a) combining the protein of claim 1with the library of molecules or compounds under conditions to allowspecific binding; and (b) detecting specific binding, therebyidentifying a molecule or compound which specifically binds the protein.